Herpes simplex virus type 2 (HSV-2) is the major cause of genital herpes, one of the most frequent sexually transmitted diseases. The worldwide prevalence of genital HSV-2 continues to increase and the limited success of HSV-2 glycoprotein subunit vaccines underscores the urgency of defining innate resistance and protective immune responses to HSV-2 in humans. Because of the high seropositivity rate of HSV throughout the world, evidence of innate or acquired resistance to HSV was not previously suspected, We have, however, in the last year identified a group of individuals who are seronegative to HSV by repeated analyses using the most sensitive serologic assays but who possess CD4+ and CD8+ T cell responses to HSV at multiple time points over the course of prospective follow-up. Preliminary study revealed no evidence of HSV infection in these subjects. The goal of this proposal is to determine whether HSV-seronegative subjects who are chronically exposed to HSV-2 from infected partners exhibit acquired and innate mechanisms of resistance to HSV infection. These subjects are classified as immune seronegative, IS, if they possess HSV-specific T cell responses or as exposed-seronegative, ES, if they do not possess HSV-specific T cell responses. Specific Aim #I will identify IS subjects from HSV-2 discordant couples and evaluate if HSV-specific T cell responses differ qualitatively or quantitatively from those observed in HSV-infected persons with frequently reactivating genital herpes. We will characterize systemic and local T cell responses to HSV using standard chromium release assays, Elispot, intracellular cytokine staining and tetramer analysis. We will also determine if local antibody responses develop in ES and IS subjects. Specific Aim #2 will determine if polymorphisms exist in 3 HSV entry receptor genes, HVEM, nectin-1, and nectin-2 to evaluate whether one mechanism of resistance to HSV-2 infection could be analogous to the receptor mutations detected in some HIV-1-resistant persons. All 3 genes will be sequenced from ES and IS subjects and relevant HSV-2 infected subjects. If coding polymorphisms are present, we will determine whether these changes alter the efficiency of viral entry. Specific Aim #3 will explore a role for CD8-derived chemokines, MIP-lalpha, MIP-1beta and RANTES, in resistance to HSV infection. Preliminary data suggest that these chemokines are secreted at higher levels in IS subjects compared to non-IS subjects and further, that MIP-la inhibits HSV infection. We will determine if Beta-chemokines inhibit the binding of HSV to cell surface glycosaminoglycans, which binding is know to facilitate HSV entry. The results of these studies will improve our understanding of effective immune defense against HSV-2 infection and may identify a mechanism of genetic resistance to HSV. The data generated will be relevant to designing and evaluating strategies for HSV-2 preventative vaccines and immunotherapy.